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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Edition 11
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Celanese Corporation
- Date received: 11 July 1986

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: responsdibility of the sponsor, given
- Solubility and stability of the test substance in the solvent/vehicle: miscible in water, solubility tested in McCoy's 5a culture medium at 500 mg/mL (pH 10)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: 1:100 in to culture medium, 5.0 mg/mL (pH 7.5)

FORM AS APPLIED IN THE TEST solution within McCoy's 5a culture medium

OTHER SPECIFICS:
- measurement of pH in the culture medium to which the test chemical is added:
- stock solution (500 mg/mL): pH 10
- final dilution (5.0 mg/mL): ph 7.5

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-WBL
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Dr. S. Wolff, University of California, Sa. Francisco
- Normal cell cycle time (negative control): 12 - 14h

For cell lines:
- Absence of Mycoplasma contamination: examination of slides, stained with Hoechst 33258 under UV microscopy as well as decrease uptake of BrdUrd in cell cycle delay test
- Number of passages if applicable: 5-8
- Cell cycle length: 12 - 14h
- Modal number of chromosomes: 21 per cell
- Periodically checked for karyotype stability: no

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: McCoy's 5a medium, supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin and streptomycin
Cytokinesis block (if used):
0.1 µg/mL Colcemid was added to cultures (in standard cell culture medium) after washing
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : male Sprague Dawley rat liver
- method of preparation of S9 mix: derived from rats were treated with Arocolor 1254 inducing mixed function of oxidase enzymes
Test concentrations with justification for top dose:
Based on a rangefinding assay a 20h harvest time was chosen for the aberration assay with concentrations ranging from 37.5 µg/mL through 500 µg/mL.


Rangefinding assay:
- without metabolic activation:
- with a test item concentration of 16.7, 50, 167, 500 µg/mL and 1.67, 5.0 mg/mL, precipitate was present
- toxicity in the monolayer was observed at 500 µg/mL through 5.0 mg/mL
- no mitotic cells available at 1.67 and 5.0 mg/mL
- first mitotic division observed at 167 µg/mL and 500 µg/mL; cell cycle delay observed through 16.7 µg/mL
- with metabolic activation.
- precipitate present, toxicity to monolayer, mitotic cells and first mitotic division comparable to testing without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: McCoy's 5a culture medium

- Justification for choice of solvent/vehicle: solubility of test item was given in culture medium
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1 (and dose range finding)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1.2 * 10E6
- Test substance added in suspension

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: for testing with metabolic activation: 2h hour exposure period, followed by washing and 17.8h incubation
- Exposure duration/duration of treatment: 17.2h for non-metabolic activation testing, 2 + 17.8h for metabolic activation testing
- Harvest time after the end of treatment (sampling/recovery times): 2.5h after washing step

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colcemide (colchicine analoga) with 0.1 µg/mL for 2.5h before harvesting of the cells
- Methods of slide preparation and staining technique used including the stain used: good morphology cells were selected and cells with number of centromers equal to modal number of 21 +/- 2 (range 19-23) were air dried, stained in pH 6.8 buffered 5% Giemsa
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 25-100 cells from each duplicate (for positive control at least 25 cells; 100 cells for test item and negative/solvent control)
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- not computed: chromatid gap, chromosome gap, uncoiled gap, polyploid cell, endoreduplication
- simple computed: chromatid break, chromosome break, double minute fragment
- complex computed: interstitial deletion, triradial, quadriradial, complex rearrangement, dicentric, tricentric, multicentric, ring, ringchromatid, chromosome interchange, translocation, pulverised chromsome
- Determination of polyploidy: A cell containing multiple copies of the number (n) of chromosomes. Only indexed if very common. Not counted in the cells, scored for aberrations.
- Determination of endoreplication: 4n cell in which separation of chromosome pairs has failed. Only indexed if very common. Not counted in the cells scored for aberrations.
Evaluation criteria:
1. The overall chromosomal aberration frequencies.
2. The percentage of cells with any aberrations.
3. The percentage of cells with more than one aberration.
4. Any evidence for increasing amounts of damage with increasing dose, i.e., a positive dose response.
5. The estimated number of breaks involved in the production of the different types of aberrations which were observed, i.e., complex aberrations may have more significance than simple breaks.
Statistics:
Fisher's exact test: percentage of cells with aberrations in each treatment group in comparison with the results from the pooled solvent and negative controls
- significance established: p<0.05

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- precipitation observed at 1.67 mg/mL and 5.0 mg/mL at the time of the harvest
- citotoxicity observed at 500 µg/mL through 5.0 mg/mL
- first mitotic division cells obtained at 167 µg/mL and 500 µg/mL; cell cycle delay obsorved through 16.7 µg/mL

Any other information on results incl. tables

The following abbreviations are used in the tables 1-4:

TG: Chromatid gap, SG: Chromosome gap, TB: Chromatide break, SB: Chromosome break, DM: "Souble Minute" fragment, ID: Interstitial deletion, TR: triradial (three-armed cofiguration of a chromosome), QR: Quadriradial, CR: Complex rearrangement, D: Diecentric, R: Ring, CI: Chromsome Interchange, PU: Pulverized chromosome, GT: Greater than 10 aberrations

Table 1: Results of the chromosome aberration without activation, cells fixed 20.1 hours after treatment

Treatment Cells sorted Number and type of aberration No. of aberrations per cell % cells with aberrations % cells with >1 aberrations
Not computed Simple Complex Other
TG SG TB SB DM ID TR QR CR D R CI PU GT
Controls
 Negative and solvent
200 5 2 1 2 0.03 2.5 0
 Positive: Mitomycin C
 80 ng/mL
25 1 1 1 4 2 0.32 24.0* 8.0*
Test compound
50 µg/mL 100 5 20 20 6 35 32 2 4 7 1 1 >1.37 77.0* 35.0*
125 µg/mL 50 2 2 33 12 7 31 15 9 2 7 2 7 >3.76 96.0* 78.0*
250 µg/mL 50 2 2 25 38 6 30 12 9 5 22 >6.90 100.0* 98.0*
375 µg/mL 50 14 1 30 33 4 31 20 14 2 1 6 17 >6.22 100.0* 100.0*
500 µg/mL (a)

(a) chromosome morphology too degenerate for accurate analysis.

* Significantly greater than the pooled negative and solvent controls, p<0.01

Table 2: Results of the chromosome aberration with activation, cells fixed 20.1 hours after treatment

Treatment Cells sorted Number and type of aberration No. of aberrations per cell % cells with aberrations % cells with >1 aberrations
Not computed Simple Complex Other
TG SG TB SB DM ID TR QR CR D R CI PU GT
Controls
 Negative and solvent
200 2 0 0 0
 Positive: Cyclophosphamide
 12.5 µg/mL
25 1 2 1 2 4 1 1 0.44 32.0* 12.0*
Test compound
50 µg/mL 200 12 5 4 18 10 2 1 3 0.19 13.0* 3.5
125 µg/mL 200 17 5 23 25 3 28 16 3 2 2 3 0.53 35.5* 12.5*
250 µg/mL 50 1 2 34 24 7 25 16 4 6 2 8 >3.96 100.0* 88.0*
375 µg/mL 50 5 1 34 44 11 26 18 9 1 5 8 4 7 >4.60 100.0* 90.0*
500 µg/mL (a)

(a) Toxic level

* Significantly greater than the pooled negative and solvent controls, p<0.01

Applicant's summary and conclusion

Conclusions:
The test item is considered positive for inducing chromosomal aberrations in Chinese hamster ovary cells under both the metabolic activation and nonactivation conditions of the performed assay.